This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of the research is to examine the role that protons (H+) may play in the processing of signals in the outer retina. Our specific aims are (a) to measure activity-dependent changes in H+ concentration in the invaginating synapse of cone photoreceptors of the zebrafish, and (b) to examine "microdomains" of proton flux from single horizontal cells isolated from the retina of the skate. The proposed research is a collaborative endeavor undertaken by Richard Kramer from the University of California at Berkeley and Robert Paul Malchow from the University of Illinois at Chicago. Studies examining certain characteristics of proton flux from isolated horizontal cells from both skate and catfish have already been done in collaboration with BRC personnel and using facilities at the BRC. Thus far, we have been able to show that application of glutamate as well as direct depolarization via voltage clamp leads to an alkalinization of the extracellular surface of isolated horizontal cells through the activation of a PMCA-pump. This work has been published in the Journal of Physiology (Molina et al. 2004) and the Journal of General Physiology (Kreitzer et al. 2007). We plan to examine changes in proton flux at two locations from horizontal cells isolated from the retina of the skate. We will use focal UV stimulation to uncage glutamate next to one arm of a horizontal cell while measuring proton flux at two locations using self-referencing electrodes. We also plan to examine fluorescent signals in the intact retina from a GFP-tagged glutamate receptor that should be expressed specifically in horizontal cells of the zebrafish. The fluorescent signals generated by this compound are highly sensitive to changes in extracellular H+ concentration. Thus, we hope to be able to make measurements of H+ concentration changes in the intact retina in a highly defined area near the site where alterations in proton flux have been hypothesized to play a key role in the shaping of visual signals. We anticipate using the two-photon microscope available at the MBL to make many of these measurements.